Fig. 7.

Impeding UCKL1-SLC7A11 axis synergizes with GPX4 inhibitor in ferroptosis induction and tumour growth suppression. (a) Cell viabilities of HCT116 cells treated with UCKL1 knockdown or/and RSL3. For the combined treatment, cancer cells were pretreated with doxycycline for 2 days, and then treated with 2 μM RSL3 for 24 h. One-way ANOVA was used to compare the results. (b) Cell viabilities of HCT116 cells treated with UCKL1 knockdown or/and ML162. For the combined treatment, cancer cells were pretreated with doxycycline for 2 days, and then treated with 5 μM ML162 for 24 h. One-way ANOVA was used to compare the results. (c) Lipid peroxidation level of UCKL1 shRNA transduced HCT116 cells treated with doxycycline or/and RSL3. Cancer cells were pretreated with doxycycline for 3 days, and then treated with 5 μM RSL3 for 4 h. One-way ANOVA was used to compare the results. (d) Lipid peroxidation level of UCKL1 shRNA transduced HCT116 cells treated with doxycycline or/and ML162. Cancer cells were pretreated with doxycycline for 3 days, and then treated with 10 μM ML162 for 4 h. One-way ANOVA was used to compare the results. (e) Relative ROS level of UCKL1 shRNA transduced HCT116 cells treated with doxycycline or/and RSL3. Cancer cells were pretreated with doxycycline for 3 days, and then treated with 5 μM RSL3 for 4 h. One-way ANOVA was used to compare the results. (f) Relative ROS level of UCKL1 shRNA transduced HCT116 cells treated with doxycycline or/and ML162. Cancer cells were pretreated with doxycycline for 3 days, and then treated with 10 μM ML162 for 4 h. One-way ANOVA was used to compare the results. (g) Volume of HCT116 xenografts treated with UCKL1 knockdown or/and RSL3 (50 mg/kg) at different time points (n = 5). Drinking water containing doxycycline (2 mg/ml) was used to induce UCKL1 knockdown. RSL3 was injected intratumourally twice per week. Tumour size was measured every 3 days and calculated according to the equation volume = length × width² × 1/2. Two-way ANOVA was used for comparison of differences between growth curves. (h) Tumour weights of HCT116 xenografts treated with doxycycline or/and RSL3 at the end point. One-way ANOVA was used to compare the result. (i) Representative images of HCT116 xenografts treated with doxycycline or/and RSL3 at the end point. (j) Representative real-time qPCR result of UCKL1 mRNA level in one of the tumour tissues of indicated mouse group. One-way ANOVA was used to compare the result. (k) Representative real-time qPCR result of SLC7A11 mRNA level in one of the tumour tissues of indicated group. One-way ANOVA was used to compare the result. (l) Representative real-time qPCR result of PTGS2 mRNA level in one of the tumour tissues of indicated group. One-way ANOVA was used to compare the result. (m) Representative images of haematoxylin and eosin staining and IHC staining of UCKL1, SLC7A11, and 4-HNE in the tumour tissues of indicated group. Scale bar: 25 μm.